Abstract 5-Methylation (m5C) on mRNA molecules is a prevalent internal posttranscriptional modification in eukaryotes. Although m5C modification has been reported to regulate some biological processes, whether most mRNA m5C modifications are functional is unknown. To address this question, we analyzed the genome-wide evolutionary characteristics of m5C modifications in protein-coding genes of humans and mice. Our analysis of RNA sequencing data from 13 tissues of both species revealed that (i) the occurrence of m5C modification is exceedingly low, (ii) the fraction of m5Cs decreases with the amount of Cs across genes or tissues, (iii) m5C modifications are mostly unshared between species, and (iv) m5C sites and motifs do not exhibit greater evolutionary conservation. Additionally, we estimate that a large fraction of the observed mRNA m5C modifications may be deleterious. Together, these observations suggest that most m5C modifications in mammalian mRNAs are nonadaptive, which has important implications for understanding the biological significance of m5C and other posttranscriptional modifications.